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Introduction
Introduction
In this chapter we will again be building on the central dogma of molecular biology as in chapter 5. However, while we examined the effects of differentiation on RNA before, we will now look at the effects of differentiation on protein actvity (specifically of gelatinases) produced by untreated and treated cells.
Matrix metalloproteases function as the name implies, they break down proteins in the extracellular matrix. This enables various leukocytes (white blood cells of our immune system) to move through the extracellular matrix by degrading proteins which may be blocking movement. The extracellular matrix acts as a web of supporting structures to keep cells and tissues in place. When a leukocyte needs to move to another area, it releases MMPs in order to create a path through the surrounding matrix. Remember that specific leukocytes (such as macrophages) function by ingesting invading pathogens and other foreign material, so it is critical that the cell be able to move to areas of infection.
The substrates of MMP-9 are: collagen, gelatin, elastin, pro-TNF-a, and interleukins. In today’s experiment, we will analyze gelatinase activity from untreated, DMSO, and PMA-treated HL-60 cells. Last time we isolated RNA to look at the cell products, this time we will analyze the activity of the end product itself, the protein. In the next section of this chapter we will be using HL-60 cells, both PMA treated and DMSO treated (as well as untreated) to examine enzymatic activity of protein isolates using a zymography electrophoresis gel. By providing gelatin as a substrate, we should be able to visualize the cell types that had functioning MMP-9.
Figure 1. The central dogma of molecular biology is the principle of directional flow of genetic information proceeds from DNA to RNA to protein. The flow of information involves DNA replication, transcription (copying information without “language” change) from DNA to RNA, and translation (language change from nucleotide sequence to amino acid sequence) from RNA to protein. Genetic expression of the eukaryotic cell is reflected in the products produced, i.e., RNA or proteins. Control of this genetic expression is exerted at different levels during this process: 1) genomic, 2) during transcription, 3) during RNA processing, 4) during translation, and 5) posttranslationally
SDS-PAGE
SDS-PAGE
Electrophoresis is a technique that has been used previously in this laboratory. Remember that molecules can be separated on the basis of size and charge using gel electrophoresis. However, the methods for separating proteins are a little different than the methods we employed to separate DNA molecules.
To separate proteins, polyacrylamide gels are typically used rather than agarose. These gels provide much better resolution of molecular separation than agarose, but the principle is still the same. Small molecules move through the matrix very quickly but larger molecules move slowly.
Also, the samples are prepared in a buffer that contains SDS (Sodium Dodecyl Sulfate). This molecule is an anionic detergent (negatively charged), and it is also amphipathic (it contains both hydrophobic and hydrophilic regions). SDS is used in protein electrophoresis to denature secondary and non-disulfide tertiary structures (so, the proteins lose their native shape). These characteristics enable SDS to interact with all parts of a protein, allowing the protein to take on the negative charge of the SDS, so all of the proteins will migrate towards the positive electrode. (Remember, proteins are composed of amino acids with have various properties including positive and negative charges depending on pH, SDS helps combat that).
The buffer that is used to load the sample generally contains dyes that allow us to follow the protein migration through the gel, such as Bromophenol Blue. It also typically contains glycerol (to make the protein sample more dense than the buffer so it stays in the well of the gel), β-Mercaptoethanol (which degrades sulfhydryl bonds to denature the proteins), and SDS (which neutralizes the native charges of the protein). Also, high heat is often used to further denature the proteins, so that the shape of the proteins does not affect their migration rate.
If we were just analyzing the protein content of our samples, we could perform an SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Eletrophoresis). This method simply separate all the proteins in our cell samples by size and charge. To visualize them, we must stain with a specific molecule. One of the most used is Coomassie Brilliant Blue which binds to basic amino acids typically and allows for the appearance of a blue band where proteins are located. Generally, the larger the band, the more abundant the protein. Note that SDS-PAGE is great for general protein content, but it cannot isolate specific proteins (that's typically done in a follow method).
Figure 2. Sodium Dodecyl Sulfate Electrophoresis Gel apparatus. Two glass plates are separated by a spacer. The bottom end rests in a buffer in which lies the positive electrode. Negative electrode lies in a buffer at the top end where the stacking gel with the wells to load samples is provided. The running gel below the stacking gel settles small proteins down and big proteins at the top.
Figure 3. PMA Signaling Pathway: Emphasis protein expression/activity. PMA binds to specific receptors embedded in the cell membrane to induce the production of secondary messengers such as calcium to activate the kinase PKC-β which in turn phosphorylates and activates a trancription factor that induces the expression of MMP-9 mRNA. That mRNA is then exported from the nucleus (after processing) and is translated into the pro-enzyme MMP9. The proenzyme can then be exported from the cell and processed to give rise to the fully active MMP-9 proteinase. Image courtesy of Christopher Malloy.
Zymogram Gel Analysis of MMP-9 Proteolytic Activity
Zymogram Gel Analysis of MMP-9 Proteolytic Activity
For this exercise we will use HL-60 cells that have been treated with either PMA or DMSO (along with untreated cells as a control). We will be observing enzymatic activity of two enzymes: MMP-9, and MMP-2. MMP-2 is another member of the matrix metalloproteinase family, used by both monocytes and granuloctyes. The function of MMP-2 is similar to MMP-9 in that it degrades the extracellular matrix to allow for motility. MMP-9 however, is specific to monocytes. MMP-9 and MMP-2, are not enzymatically active until they leave the cell and a pro-peptide sequence is cleaved from the N-terminal end. Secreted enzymes are located in the growth medium, so rather than collecting protein extracts, we will be running medium containing enzymatically active MMP-9 on a different type of gel, a zymogram gel.
A zymogram gel is a polyacrylamide gel that has been infused with gelatin (remember, this is a substrate of MMP-9 as well as MMP-2, they are gelatinases). We know that in order for a molecule to exert enzymatic activity, it cannot be denatured. However, for a protein to migrate through a gel towards the positive electrode, we must denature it! To solve this dilemma, we treat our zymography samples with chemicals which denature the proteins only temporarily- we will renature the proteins after running the zymogram. If MMP-9 and MMP-2 are enzymatically active, they will act on the gelatin in the polyacrylamide gel. We will stain our entire gel blue using Coomasie Blue stain after our proteins have renatured. Wherever MMPs have degraded the gelatin will appear white on the blue gel (Figure 4).
Example Results
Example Results
Figure 4. Example Zymogram. Here is an example of a zymogram that was run in class. Lane 1 and 2 are from untreated HL60 cells, Lanes 3 and 4 are from PMA treated HL60 cells, and Lanes 5 and 6 are from DMSO treated HL60 cells. The enzymatic activity of MMP-9 can be noticed at a higher extent in the PMA treated cells (the clearer the band, the more enzyme activity). This is because PMA causes HL-60 cells to differentiate into monocytes which, in turn, transcribe the MMP-9 gene which is translated into MMP-9 protein. The reason the band is clear is because the MMP-9 is actively cleaving its substrate (gelatin) which is present in the gel. We don’t see as much MMP-9 activity in the DMSO treated cells because they are incapable of producing the MMP-9 protein, so substrate cleavage is reduced. Remember that there is a mixed population of cell types in every treatment group because of how easily HL-60 cells can be induced to differentiate. So even though we have a DMSO treatment group, we are still seeing a small amount of MMP-9 activity in that treatment group because some of the HL-60 cells are spontaneously differentiating into monocytes, thereby producing a small amount of MMP-9 protein. Remember, MMP’s are not rendered active until they leave the cell and a pro-peptide sequence is cleaved from the N-terminus. Therefore, we did NOT need to collect whole cell extracts since no active MMP’s are found inside the cell. The active enzymes are secreted into the growth medium and can be collected in the supernatant after whole cell centrifugation. MMP-2 is basically used as a control (much like B-actin in the RT-PCR experiment) because it is active in both granulocytes and monocytes. It can be used as a control to let us know that active MMP’s are being cleaved in the growth medium and that the zymography gel electrophoresis worked properly.
Exercise 1: Gelatin Zymography
Exercise 1: Gelatin Zymography
Glossary
Glossary
1. Amphipathic: a chemical compound possessing both hydrophilic and hydrophobic groups
2. Anionic: ionic group with a negative charge
3. Bromophenol blue: blue dye that has a slightly negative charge, and is often included in loading buffers during electrophoresis to indicate the progression of the sample
4. Electrophoresis: method of separation of biological molecules based on their size and charge
5. Extracellular matrix: a protein scaffold existing in the intercellular spaces which connects cells to one another and provides support
6. Gelatinase: enzyme that catalyzes the hydrolysis of gelatin, a type of collagen molecule often present in the extracellular matrix
7. Glycerol: very viscous organic compound used as a thickening agent or mild sweetener
8. Matrix metalloproteases: cellular enzymes that degrade extracellular matrix proteins, and other molecules; a major contributor to cell motility
9. Polyacrylamide gels: separation matrix that provides a greater resolution than agarose matrix, allowing for separation of proteins or very small DNA molecules
10. SDS-PAGE: polyacrylamide gel electrophorsis utilizing sds denaturation of proteins
11. Sodium Dodecyl Sulfate: an anionic and amphipathic molecule that binds to hydrophic and hydrophilic portions of proteins in order to impart an overall negative charge
12. ß-Mercaptoethanol: chemical that cleaves disulfide bonds to disrupt tertiatry and quaternary structure of proteins
13. Substrates: the molecule upon which an enzyme acts
14. Zymography: type of SDS-PAGE that includes a substrate copolymerized with the polyacrylamide to detect the level of enzymatic activity of certain proteins
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